Human cytomegalovirus neutralising antibodies and use thereof

ABSTRACT

The invention relates to neutralizing antibodies and antibody fragments having high potency in neutralizing hCMV, wherein said antibodies and antibody fragments are specific for a combination of hCMV proteins UL130 and UL131A, or for a combination of hCMV proteins UL128, UL130 and UL131A. The invention relates also to immortalized B cells that produce, and to epitopes that bind to, such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies, antibody fragments, and epitopes in screening methods as well as in the diagnosis and therapy of disease.

This application is a continuation of U.S. patent application Ser. No.13/618,264, filed Sep. 14, 2012, which is a continuation of U.S. patentapplication Ser. No. 13/087,814, filed Apr. 15, 2011, which is acontinuation of U.S. patent application Ser. No. 12/174,568 filed Jul.16, 2008, which is a continuation in part of U.S. patent applicationSer. No. 11/969,104, filed Jan. 3, 2008, which claims priority toBritish Patent Application No. GB 0700133.2, filed Jan. 4, 2007. Theentire contents of each of these patents and patent applications, alongwith all documents cited therein, are hereby incorporated herein byreference.

BACKGROUND

This invention relates to potent, neutralizing antibodies havingspecificity for human cytomegalovirus, and immortalized B cells thatproduce such monoclonal antibodies. The invention also relates to theepitopes that the antibodies bind to as well as the use of theantibodies and the epitopes in screening methods as well as thediagnosis, prophylaxis and therapy of disease.

Human cytomegalovirus (hCMV) is a widely distributed pathogen that maycause severe pathology in immunosuppressed adults and upon infection ofthe fetus and has been implicated in chronic diseases such asatherosclerosis. hCMV infects multiple cell types including fibroblasts,endothelial, epithelial and hematopoietic cells [1]. In vitro propagatedattenuated strains of hCMV, which are being developed as candidatevaccines, have lost the tropism for endothelial cells, while retainingthe capacity to infect fibroblasts [2]. Two viral glycoprotein complexesare believed to control the cellular tropism of hCMV. A complex of gH,gL and gO is required for infection of fibroblasts, while a complex ofgH, gL and proteins encoded by the UL131-UL128 genes are responsible forinfection of endothelial cells, epithelial cells and dendritic cells[2-8].

Hyperimmune globulins are already commercialized for the prophylaxis ofhCMV disease associated with transplantation and recent evidenceindicates that they have therapeutic effect in pregnant women [9]. Thistherapeutic approach is limited by the low amount of neutralizingantibody that can be transferred and for this reason the availability ofhuman antibodies (such as human monoclonal antibodies) with highneutralizing capacity would be highly desirable. However the target ofhCMV neutralizing antibodies remains to be established. Although someantibodies to gH, gB and UL128 and UL130 gene products have demonstratedin vitro neutralizing activities [7, 10, 11] and an antibody to gH hasbeen evaluated in clinical trials which were discontinued due to lack oftherapeutic effects, the neutralizing potency of the monoclonalantibodies isolated so far is modest, since neutralization was observedat antibody concentrations ranging from 0.5 to 20 microgram/ml. Further,the current methods typically measure the neutralising potency ofanti-hCMV antibodies using fibroblasts as target cells. However, hCMV isalso known to cause pathology by infecting other cell types such asendothelial, epithelial cells and leukocytes. Known antibodies to UL128and UL130 show very low potency in neutralising infection of endothelialcells [7] and there do not appear to be any monoclonal antibodiesavailable that would be capable of neutralising infection ofnon-fibroblast target cells with high potency.

There is therefore a need for the production of neutralizing antibodiesagainst hCMV infection of non-fibroblast cells as well as theelucidation of the target to which such antibodies bind.

SUMMARY OF INVENTION

The invention is based, in part, on the discovery of novel antibodiesthat neutralize hCMV with high potency as well as novel epitopes towhich the antibodies of the invention bind. Accordingly, in oneembodiment, the invention comprises a neutralizing antibody and antibodyfragments having high potency in neutralizing hCMV, wherein saidantibody and antibody fragments are specific for a combination of hCMVproteins UL130 and UL131A, or for a combination of hCMV proteins UL128,UL130 and UL131A.

In another embodiment the invention comprises a nucleic acid moleculeencoding an antibody or an antibody fragment of the invention.

In yet another embodiment the invention comprises a vector comprising anucleic acid molecule encoding an antibody or an antibody fragment ofthe invention.

In a further embodiment the invention comprises a cell comprising avector comprising a nucleic acid molecule of the invention.

In another embodiment the invention comprises an immortalized B cellclone expressing an antibody of the invention.

In yet another embodiment the invention comprises an epitope which bindsto an antibody of the invention.

In a further embodiment the invention comprises an immunogenicpolypeptide comprising an epitope which binds to an antibody of theinvention.

In another embodiment the invention comprises a ligand which binds to anepitope which binds to an antibody of the invention.

In a further embodiment the invention comprises a method for producingantibodies having high potency in neutralizing hCMV, wherein saidantibody is specific for a combination of the hCMV proteins UL128, UL130and UL131A or for a combination of the hCMV proteins UL130 and UL131A.The method comprises culturing an immortalized B cell clone expressingan antibody of the invention and isolating antibodies from the B cell.

In another embodiment the invention comprises a pharmaceuticalcomposition comprising an antibody or antibody fragment, a nucleic acidof the invention, an immortalized B cell clone expressing an antibody ofthe invention, or an immunogenic polypeptide comprising an epitope whichbinds to an antibody of the invention.

In a further embodiment the invention comprises an antibody or antibodyfragment having high potency in neutralizing hCMV, wherein said antibodyor antibody fragment is specific for a combination of the hCMV proteinsUL130 and UL131A or for a combination of the hCMV proteins UL128, UL130,UL131A, a nucleic acid encoding an antibody or an antibody fragment asabove, an immortalized B cell clone expressing an antibody having highpotency in neutralizing hCMV wherein said antibody is specific for acombination of the hCMV proteins UL130 and UL131A or for a combinationof the hCMV proteins UL128, UL130 and UL131A, or an immunogenicpolypeptide comprising an epitope which binds to an antibody or antibodyfragment having high potency in neutralizing hCMV, wherein said antibodyis specific for a combination of the hCMV proteins UL130 and UL131A orfor a combination of the hCMV proteins UL128, UL130 and UL131A for usein therapy or diagnosis.

In another embodiment the invention comprises a kit for the diagnosis ofhCMV infection comprising antibodies or antibody fragments of theinvention, or a nucleic acid encoding an antibody or an antibodyfragment of the invention.

In another embodiment the invention comprises a method for preparing arecombinant cell. The method comprises sequencing nucleic acid from animmortalized B cell clone expressing an antibody of the invention andusing the sequence information to prepare nucleic acid for insertinginto an expression host in order to permit expression of the antibody ofinterest in that host.

In a further embodiment the invention comprises a method for producingantibodies having high potency in neutralizing hCMV and specific for acombination of the hCMV proteins UL128, UL130 and UL131A or for acombination of the hCMV proteins UL130 and UL131A. The method comprisesculturing or sub-culturing an expression host obtainable by the methoddescribed above and, optionally, purifying the antibody of interest.

In another embodiment the invention comprises a method of screening forpolypeptides that can induce an immune response against hCMV, comprisingscreening polypeptide libraries using an antibody or an antibodyfragment of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a SDS-PAGE which demonstrates that human monoclonalantibodies (1) 1F11 and (2) 2F4 precipitate complexes of hCMV proteins,whereas irrelevant IgG does not.

FIG. 2 shows a FACS analysis which demonstrates that human monoclonalantibodies (A) 1F11 and 2F4 and (B) 5A2 and 9A11 recognize an epitopedetermined by a combination of the hCMV proteins UL and UL131A.

FIG. 3 shows the nucleotide and amino acid sequences of the variableregions of the heavy and light chains of antibodies 1F11 (SEQ ID NOs: 9and 10 and SEQ ID NOs: land 8, respectively) and 2F4(SEQ ID NOs: 19 and20 and SEQ ID NOs: 17 and 18, respectively). The CDR sequences are inbold.

FIG. 4 shows a FACS analysis which demonstrates that the humanmonoclonal antibodies 7H3, 1006, 5F1 and 6B4 recognize an epitope onhCMV protein gB.

FIG. 5 shows the nucleotide and amino acid sequences of the variableregions of the heavy and light chains of antibody 5A2 (SEQ ID NOs:41 and42 and SEQ ID NOs: 39 and 40, respectively). The CDR sequences are inbold.

FIG. 6 shows FACS analysis which demonstrates that the human monoclonalantibody 6G4 recognizes an epitope determined by the hCMV proteinsUL128, UL130 and UL131A.

FIG. 7 shows the nucleotide and amino acid sequences of the variableregions of the heavy and light chains of antibody 6G4 (SEQ ID NOs: 65and 66 and SEQ ID NOs: 63 and 64, respectively). The CDR sequences arein bold.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the production of antibodies and antibodyfragments that neutralize hCMV infection and which have a particularlyhigh potency in neutralizing hCMV infection. Such antibodies aredesirable, as only low concentrations are required in order toneutralize a given amount of virus. This facilitates higher levels ofprotection whilst administering lower amounts of antibody. Humanmonoclonal antibodies and the immortalized B cell clones that secretesuch antibodies are also included within the scope of the invention.

The inventors have discovered that antibodies directed to a combinationof UL130 and UL131A are particularly effective in neutralizing hCMV. Thecombination may be a complex of UL130 and UL131A forming an epitoperecognized by the antibody or an antibody may be directed to one ofUL130 and UL131A, the presence of the other protein being necessary forspecificity. In addition, the inventors have discovered that antibodiesdirected to a combination of UL128, UL130 and UL131A are particularlyeffective in neutralizing hCMV. Without being bound to any theory, thiscombination may be a precise complex of UL128, UL130 and UL131A forminga unique epitope recognized by the antibody.

The invention also relates to the characterization of the epitope towhich the antibodies bind and the use of that epitope in raising animmune response.

The invention also relates to various methods and uses involving theantibodies of the invention and the epitopes to which they bind.

Antibodies

The invention provides monoclonal or recombinant antibodies havingparticularly high potency in neutralizing hCMV. The invention alsoprovides fragments of these recombinant or monoclonal antibodies,particularly fragments that retain the antigen-binding activity of theantibodies, for example which retain at least one complementaritydetermining region (CDR) specific for a combination of hCMV proteinsUL130 and UL131A or retain at least one CDR specific for a combinationof the hCMV proteins UL128, UL130 and UL131A.

In this specification, by “high potency in neutralizing hCMV” is meantthat an antibody molecule of the invention neutralizes hCMV in astandard assay at a concentration much lower than antibodies known inthe art, for example compared to MSL-109, 8F9 or 3E3. For example, theterm “high potency” is used to refer to an antibody or an antibodyfragment of the invention that has IC₅₀ of less than about 2 μg/ml,(i.e. the concentration of antibody required for 50% neutralization of aclinical isolate of hCMV is about 2 μg/ml or less).

In one embodiment, the antibody or antibody fragment of the presentinvention can neutralise hCMV at a concentration of 1 μg/ml or less(i.e. 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1,0.05, 0.01 μg/ml or lower). In another embodiment, the antibody orantibody fragment of the present invention can neutralise hCMV at aconcentration of 0.16 μg/ml or lower (i.e. 0.15, 0.125, 0.1, 0.075,0.05, 0.025, 0.02, 0.015, 0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003,0.002 μg/ml or lower). In another embodiment, the antibody canneutralise hCMV at a concentration of 0.016 μg/ml or lower (i.e. at0.015, 0.013, 0.01, 0.008, 0.005, 0.003, 0.001, 0.0005 μg/ml or lower).This means that only very low concentrations of antibody are requiredfor 50% neutralisation of a clinical isolate of hCMV in vitro comparedto the concentration of known antibodies, e.g., MSL-109, required forneutralisation of the same titre of hCMV. Potency can be measured usinga standard neutralisation assay as known to one of skill in the art.

The antibodies of the invention are able to neutralize hCMV infection ofseveral kinds of cells. Preferably, an antibody according to theinvention prevents infection of fibroblasts or endothelial cells. Morepreferably, an antibody according to the invention prevents infection ofendothelial cells. Preferably, an antibody according to the inventionprevents infection of both fibroblasts and endothelial cells. Theantibodies of the invention preferably also prevent infection ofepithelial cells, retinal cells and dendritic cells.

These antibodies can be used as prophylactic or therapeutic agents uponappropriate formulation, or as a diagnostic tool.

A “neutralizing antibody” is one that can neutralize the ability of thatpathogen to initiate and/or perpetuate an infection in a host. Theinvention provides a neutralizing human monoclonal antibody, wherein theantibody recognizes an antigen from hCMV.

Preferably, an antibody according to the invention has specificity for acombination of the hCMV proteins UL130 and UL131A.

Preferably an antibody according to the invention is a monoclonalantibody referred to herein as 1F11 or 2F4. These antibodies wereinitially isolated from a hCMV infected donor, and are produced by theimmortalized B cell clones referred to as 1F11 or 2F4. These antibodieshave been shown to neutralize hCMV infection of endothelial cells,epithelial cells, retinal cells and dendritic cells. In addition, theantibodies 5A2 and 9A11, isolated from a different hCMV infected donor,show the same specificity for a combination of UL130 and UL131A and theability to neutralize hCMV infection of endothelial cells, epithelialcells, retinal cells and dendritic cells. These antibodies are producedby the immortalized B cell clones referred to as 5A2 and 9A11,respectively.

1F11 consists of a heavy chain having the amino acid sequence recited inSEQ ID NO:7 and a light chain having the amino acid sequence recited inSEQ ID NO:8. 2F4 consists of a heavy chain having the amino acidsequence recited in SEQ ID NO:17 and a light chain having the amino acidsequence recited in SEQ ID NO:18. The CDRs of the antibody heavy chainsare referred to as CDRH1, CDRH2 and CDRH3, respectively. Similarly, theCDRs of the antibody light chains are referred to as CDRL1, CDRL2 andCDRL3, respectively. The position of the CDR amino acids are definedaccording to the IMGT numbering system [12, 13, 14] as: CDR1-IMGTpositions 27 to 38, CDR2-IMGT positions 56 to 65 and CDR3-IMGT positions105 to 117.

5A2 consists of a heavy chain having the amino acid sequence recited inSEQ ID NO:39 and a light chain having the amino acid sequence recited inSEQ ID NO:40.

The amino acid sequences of the CDRs of these antibodies are shown inTable 1.

TABLE 1 1F11 2F4 5A2 CDRH1 GFTFSSY GFSFNTY GGTFSSY A G V (SEQ ID (SEQ ID(SEQ ID NO: 1) NO: 11) NO: 33) CDRH2 ISFDGDN IWDDGSK IIPIFNT K M A(SEQ ID (SEQ ID (SEQ ID NO: 2) NO: 12) NO: 34) CDRH3 AREELVG ARDEGAIARDFLSG LMPPYYN MLHAMTD PMEMPGG YGLDV YGLDV YYGLDV (SEQ ID (SEQ ID(SEQ ID NO: 3) NO: 13) NO: 35) CDRL1 SSNIGNN NLGDEF QSVLYSS F (SEQ IDNNKNY (SEQ ID NO: 14) (SEQ ID NO: 4) NO: 36) CDRL2 DND QDS WAS (SEQ ID(SEQ ID (SEQ ID NO: 5) NO: 15) NO: 37) CDRL3 ETWDGSL QAWDSST QQYYSTPNPAVV AHYV IT (SEQ ID (SEQ ID (SEQ ID NO: 6) NO: 16) NO: 38)

The invention also includes an antibody comprising a heavy chaincomprising one or more (i.e. one, two or all three) heavy chain CDRsfrom 1F11 or 2F4 (SEQ ID NOs:1-3 or 11-13). Also included is an antibodycomprising a heavy chain comprising one or more (i.e. one, two or allthree) heavy chain CDRs from 5A2 (SEQ ID NOs:33-35).

Preferably an antibody according to the invention comprises a heavychain comprising (i) SEQ ID NO:1 for CDRH1, SEQ ID NO:2 for CDRH2 andSEQ ID NO:3 for CDRH3, or (ii) SEQ ID NO:11 for CDRH1, SEQ ID NO:12 forCDRH2 and SEQ ID NO:13 for CDRH3. A further preferred antibody accordingto the invention comprises a heavy chain comprising SEQ ID NO:33 forCDRH1, SEQ ID NO:34 for CDRH2 and SEQ ID NO:35 for CDRH3.

The invention also includes an antibody comprising a light chaincomprising one or more (i.e. one, two or all three) light chain CDRsfrom 1F11 or 2F4 (SEQ ID NOs:4-6 or 14-16). Also included is an antibodycomprising a light chain comprising one or more (i.e. one, two or allthree) light chain CDRs from 5A2 (SEQ ID NOs:36-38).

Preferably an antibody according to the invention comprises a lightchain comprising (i) SEQ ID NO:4 for CDRL1, SEQ ID NO:5 for CDRL2 andSEQ ID NO:6 for CDRL3, or (ii) SEQ ID NO:14 for CDRL1, SEQ ID NO:15 forCDRL2 and SEQ ID NO:16 for CDRL3. A further preferred antibody accordingto the invention comprises a light chain comprising SEQ ID NO:36 forCDRL1, SEQ ID NO:37 for CDRL2 and SEQ ID NO:38 for CDRL3.

Preferably an antibody according to the invention comprises a heavychain having the sequence recited in SEQ ID NO:7, 17 or 39. Preferablyan antibody according to the invention comprises a light chain havingthe sequence recited in SEQ ID NO:8, 18 or 40.

Hybrid antibody molecules may also exist that comprise one or more CDRsfrom 1F11 and one or more CDRs from 2F4. Preferably, such hybridantibodies comprise three CDRs from 1F11 and three CDRs from 2F4. Thus,preferred hybrid antibodies comprise i) the three light chain CDRs from1F11 and the three heavy chain CDRs from 2F4, or ii) the three heavychain CDRs from 1F11 and the three light chain CDRs from 2F4. In analternative, such hybrids may contain one or more CDRs from 5A2.

The invention also includes nucleic acid sequences encoding part or allof the light and heavy chains and CDRs of the present invention.Preferred nucleic acid sequences according to the invention include SEQID NO:9 (encoding the 1F11 heavy chain variable region), SEQ ID NO:10(encoding the 1F11 light chain variable region), SEQ ID NO:19 (encodingthe 2F4 heavy chain variable region), and SEQ ID NO:20 (encoding the 2F4light chain variable region). Preferred nucleic acid sequences encodingthe various CDRs include SEQ ID NO:21 (encoding 1F11 CDRH1), SEQ IDNO:22 (encoding 1F11 CDRH2), SEQ ID NO:23 (encoding 1F11 CDRH3), SEQ IDNO:24 (encoding 1F11 CDRL1), SEQ ID NO:25 (encoding 1F11 CDRL2), SEQ IDNO:26 (encoding 1F11 CDRL3), SEQ ID NO:27 (encoding 2F4 CDRH1), SEQ IDNO:28 (encoding 2F4 CDRH2), SEQ ID NO:29 (encoding 2F4 CDRH3), SEQ IDNO:30 (encoding 2F4 CDRL1), SEQ ID NO:31 (encoding 2F4 CDRL2) and SEQ IDNO:32 (encoding 2F4 CDRL3). Further preferred nucleic acid sequencesaccording to the invention include SEQ ID NO:41 (encoding the 5A2 heavychain variable region), SEQ ID NO:42 (encoding the 5A2 light chainvariable region), SEQ ID NO:43 (encoding 5A2 CDRH1), SEQ ID NO:44(encoding 5A2 CDRH2), SEQ ID NO:45 (encoding 5A2 CDRH3), SEQ ID NO:46(encoding 5A2 CDRL1), SEQ ID NO:47 (encoding 5A2 CDRL2), SEQ ID NO:48(encoding 5A2 CDRL3). Due to the redundancy of the genetic code,variants of these sequences will exist that encode the same amino acidsequences.

In another embodiment, an antibody according to the invention hasspecificity for a combination of UL128, UL130 and UL131A. An antibodyaccording to the invention is a monoclonal antibody referred to hereinas 6G4. This antibody, isolated from a hCMV infected donor, is producedby the immortalized B cell clone referred to as 6G4. This antibodyneutralizes hCMV infection of endothelial cells, epithelial cells,retinal cells and dendritic cells.

The heavy chain of 6G4 has the amino acid sequence recited in SEQ IDNO:63 and the light chain has the amino acid sequence recited in SEQ IDNO:64. The CDRs of the antibody heavy chains are referred to as CDRH1,CDRH2 and CDRH3, respectively. Similarly, the CDRs of the antibody lightchains are referred to as CDRL1, CDRL2 and CDRL3, respectively. Theamino acid sequences of the CDRs of this antibody are shown in Table 2.

TABLE 2 6G4 CDRH1 GYRFTSYY (SEQ ID NO: 51) CDRH2IYPGDSDI (SEQ ID NO: 52) CDRH3 ARLSLTESGDYVGAFDI (SEQ ID NO: 53) CDRL1QSLVYSDDNIF (SEQ ID NO: 54) CDRL2 KVS (SEQ ID NO: 55) CDRL3MQGRHWPPLFT (SEQ ID NO: 56)

The invention also includes an antibody comprising a heavy chaincomprising one or more (i.e. one, two or all three) heavy chain CDRsfrom 6G4 (SEQ ID NOs:51-53) or a light chain comprising one or more(i.e., one, two or all three) light chain CDRs from 6G4 (SEQ IDNOs:54-56).

In one embodiment an antibody according to the invention comprises aheavy chain comprising (i) SEQ ID NO:51 for CDRH1, SEQ ID NO:52 forCDRH2 and SEQ ID NO:53 for CDRH3.

In another embodiment the invention includes an antibody comprising alight chain comprising one or more (i.e. one, two or all three) lightchain CDRs from 6G4 (SEQ ID NOs:54-56).

In another embodiment the invention includes an antibody comprising alight chain comprising (i) SEQ ID NO:54 for CDRL1, SEQ ID NO:55 forCDRL2 and SEQ ID NO:56 for CDRL3.

In a further embodiment an antibody according to the invention hasspecificity for a combination of UL128, UL130 and UL131A and comprises aheavy chain having an amino acid sequence that is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identical to the amino acid sequence of SEQ IDNO:63 and possessing hCMV neutralizing activity. In one embodiment, anantibody according to the invention comprises a heavy chain having thesequence recited in SEQ ID NO:63 and possessing hCMV neutralizingactivity.

In another embodiment, an antibody according to the invention hasspecificity for a combination of UL128, UL130 and UL131A and comprises alight chain having an amino acid sequence that is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% identical to the amino acid sequence of SEQ IDNO:64 and possessing hCMV neutralizing activity. In yet anotherembodiment an antibody according to the invention comprises a lightchain having the sequence recited in SEQ ID NO:64 and possessing hCMVneutralizing activity.

Antibodies of the invention also include hybrid antibody molecules thatcomprise one or more CDRs from 6G4 and one or more CDRs from anotherantibody to the same epitope. In one embodiment, such hybrid antibodiescomprise three CDRs from 6G4 and three CDRs from another antibody to thesame epitope. Thus, preferred hybrid antibodies comprise i) the threelight chain CDRs from 6G4 and the three heavy chain CDRs from anotherantibody to the same epitope, or ii) the three heavy chain CDRs from 6G4and the three light chain CDRs from another antibody to the sameepitope.

In another aspect, the invention also includes nucleic acid sequencesencoding part or all of the heavy and light chains and CDRs of theantibodies of the present invention. In one embodiment, nucleic acidsequences according to the invention include nucleic acid sequenceshaving at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identity to the nucleicacid sequence of SEQ ID NO:65 or SEQ ID NO:66. In another embodiment, anucleic acid sequence of the invention has the sequence of SEQ ID NO:65(encoding the 6G4 heavy chain variable region) or SEQ ID NO:66 (encodingthe 6G4 light chain variable region). In further embodiments, nucleicacid sequences of the invention include those encoding the various CDRsinclude SEQ ID NO:57 (encoding 6G4 CDRH1), SEQ ID NO:58 (encoding 6G4CDRH2), SEQ ID NO:59 (encoding 6G4 CDRH3), SEQ ID NO:60 (encoding 6G4CDRL1), SEQ ID NO:61 (encoding 6G4 CDRL2) and SEQ ID NO:62 (encoding 6G4CDRL3).

Variant antibodies are also included within the scope of the invention.Thus, variants of the sequences recited in the application are alsoincluded within the scope of the invention. Without being bound to anytheory, such variants may arise due to the degeneracy of the geneticcode, as mentioned above. Alternatively, natural variants may beproduced due to errors in transcription or translation or be generatedby somatic mutation in vivo during the immune response or in vitro uponculturing of immortalized B cells. A variant of 2F4 is also disclosedherein. This variant comprises an additional two serine residues at theC-terminal end of the 2F4 heavy chain amino acid sequence (SEQ IDNO:17). Thus, this variant of 2F4 consists of a heavy chain having theamino acid sequence recited in SEQ ID NO:49 and a light chain having theamino acid sequence recited in SEQ ID NO:18. The nucleic acid sequenceencoding the variant heavy chain is recited in SEQ ID NO:50. Thus,antibodies comprising the 2F4 variant heavy chain (SEQ ID NO:49) areincluded within the scope of the invention.

Further variants of the antibody sequences having improved affinity maybe obtained using methods known in the art and are included within thescope of the invention. For example, amino acid substitutions may beused to obtain antibodies with further improved affinity. Alternatively,codon optimization of the nucleotide sequence may be used to improve theefficiency of translation in expression systems for the production ofthe antibody. Further, polynucleotides comprising a sequence optimizedfor antibody specificity or neutralizing activity by the application ofa directed evolution method to any of the nucleic acid sequences of theinvention are also within the scope of the invention.

Preferably, variant antibody sequences will share 70% or more (i.e. 80%,85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with thesequences recited in the application. Preferably such sequence identityis calculated with regard to the full length of the reference sequence(i.e. the sequence recited in the application). Preferably, percentageidentity, as referred to herein, is as determined using BLAST version2.1.3 using the default parameters specified by the NCBI (the NationalCenter for Biotechnology Information; http://www.ncbi.nlm.nih.gov/)[Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1].

Further included within the scope of the invention are vectors, forexample expression vectors, comprising a nucleic acid sequence accordingto the invention. Cells transformed with such vectors are also includedwithin the scope of the invention. Examples of such cells include butare not limited to, eukaryotic cells, e.g. yeast cells, animal cells orplant cells. In one embodiment the cells are mammalian, e.g. human, CHO,HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The invention also relates to monoclonal antibodies that bind to anepitope capable of binding the monoclonal antibody 1F11 or 2F4. Theinvention also relates to monoclonal antibodies that bind to an epitopecapable of binding the monoclonal antibody 5A2. In addition, theinvention relates to monoclonal antibodies that bind to an epitopecapable of binding the monoclonal antibody 6G4.

Monoclonal and recombinant antibodies are particularly useful inidentification and purification of the individual polypeptides or otherantigens against which they are directed. The antibodies of theinvention have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labeled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme. The antibodies mayalso be used for the molecular identification and characterization(epitope mapping) of antigens.

Antibodies of the invention can be coupled to a drug for delivery to atreatment site or coupled to a detectable label to facilitate imaging ofa site comprising cells of interest, such as cells infected with hCMV.Methods for coupling antibodies to drugs and detectable labels are wellknown in the art, as are methods for imaging using detectable labels.Labeled antibodies may be employed in a wide variety of assays,employing a wide variety of labels. Detection of the formation of anantibody-antigen complex between an antibody of the invention and anepitope of interest (a hCMV epitope) can be facilitated by attaching adetectable substance to the antibody. Suitable detection means includethe use of labels such as radionuclides, enzymes, coenzymes,fluorescers, chemiluminescers, chromogens, enzyme substrates orco-factors, enzyme inhibitors, prosthetic group complexes, freeradicals, particles, dyes, and the like. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material isluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H. Such labeled reagents may be used in avariety of well-known assays, such as radioimmunoassays, enzymeimmunoassays, e.g., ELISA, fluorescent immunoassays, and the like. Seefor example, references 15-18.

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, 1-131, 1-123, 1-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described in reference 19. Inaddition, linkers may be used between the labels and the antibodies ofthe invention [20]. Antibodies or, antigen-binding fragments thereof maybe directly labeled with radioactive iodine, indium, yttrium, or otherradioactive particle known in the art [21]. Treatment may consist of acombination of treatment with conjugated and nonconjugated antibodiesadministered simultaneously or subsequently [22, 23].

Antibodies of the invention may also be attached to a solid support.

Additionally, antibodies of the invention or functional antibodyfragments thereof, can be chemically modified by covalent conjugationto, for example, a polymer to increase their circulating half-life.Preferred polymers, and methods to attach them to peptides, are shown inreferences 24-27. Preferred polymers are polyoxyethylated polyols andpolyethylene glycol (PEG). PEG is soluble in water at room temperatureand has the general formula: R(O—CH₂ —CH₂)_(n)O—R where R can behydrogen, or a protective group such as an alkyl or alkanol group.Preferably, the protective group has between 1 and 8 carbons, morepreferably it is methyl. The symbol n is a positive integer, preferablybetween 1 and 1,000, more preferably between 2 and 500. The PEG has apreferred average molecular weight between 1,000 and 40,000, morepreferably between 2,000 and 20,000, most preferably between 3,000 and12,000. Preferably, PEG has at least one hydroxy group, more preferablyit is a terminal hydroxy group. It is this hydroxy group which ispreferably activated to react with a free amino group on the inhibitor.However, it will be understood that the type and amount of the reactivegroups may be varied to achieve a covalently conjugated PEG/antibody ofthe present invention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. POG ispreferred. One reason is because the glycerol backbone ofpolyoxyethylated glycerol is the same backbone occurring naturally in,for example, animals and humans in mono-, di-, triglycerides. Therefore,this branching would not necessarily be seen as a foreign agent in thebody. The POG has a preferred molecular weight in the same range as PEG.The structure for POG is shown in reference 28, and a discussion ofPOG/IL-2 conjugates is found in reference 24.

Another drug delivery system for increasing circulatory half-life is theliposome. Methods of preparing liposome delivery systems are discussedin references 29, 30 and 31. Other drug delivery systems are known inthe art and are described in, for example, references 32 and 33.

Antibodies of the invention are preferably provided in purified form.Typically, the antibody will be present in a composition that issubstantially free of other polypeptides e.g. where less than 90% (byweight), usually less than 60% and more usually less than 50% of thecomposition is made up of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g. in mice. In particular, the antibodies may havean idiotope that is immunogenic in non-human hosts, but not in a humanhost. Antibodies of the invention for human use include those thatcannot be obtained from hosts such as mice, goats, rabbits, rats,non-primate mammals, etc. and cannot be obtained by humanisation or fromxeno-mice.

Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will generally be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass.Antibodies of the invention may have a κ or a λ light chain.

Production of Antibodies

Monoclonal antibodies according to the invention can be made by one ofthe methods known in the art. The general methodology for makingmonoclonal antibodies using hybridoma technology is well known [34, 35].Preferably, the alternative EBV immortalization method described inreference 36 is used.

Using the method described in reference 36, B cells producing theantibody of the invention can be transformed with EBV in the presence ofa polyclonal B cell activator. Transformation with EBV is a standardtechnique and can easily be adapted to include polyclonal B cellactivators.

Additional stimulants of cellular growth and differentiation mayoptionally be added during the transformation step to further enhancethe efficiency. These stimulants may be cytokines such as IL-2 andIL-15. In a particularly preferred aspect, IL-2 is added during theimmortalization step to further improve the efficiency ofimmortalization, but its use is not essential.

The immortalized B cells produced using these methods can then becultured using methods known in the art and antibodies isolatedtherefrom.

Monoclonal antibodies may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Techniques for purification ofmonoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the monoclonal antibodies of the invention can be obtainedfrom the monoclonal antibodies by methods that include digestion withenzymes, such as pepsin or papain, and/or by cleavage of disulfide bondsby chemical reduction. Alternatively, fragments of the monoclonalantibodies can be obtained by cloning and expression of part ofsequences of the heavy or light chains. Antibody “fragments” includeFab, Fab′, F(ab′)₂ and Fv fragments. The invention also encompassessingle-chain Fv fragments (scFv) derived from the heavy and light chainsof a monoclonal antibody of the invention e.g. the invention includes ascFv comprising the CDRs from an antibody of the invention. Alsoincluded are heavy or light chain monomers and dimers as well as singlechain antibodies, e.g. single chain Fv in which the heavy and lightchain variable domains are joined by a peptide linker.

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibodies or fragments of the antibodies ofthe present invention. Desired DNA sequences may be synthesizedcompletely or in part using oligonucleotide synthesis techniques.Site-directed mutagenesis and polymerase chain reaction (PCR) techniquesmay be used as appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)₂ fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g. mammalian, host cell expression systems may be used forproduction of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include CHO, HEK293T, PER.C6,NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced byi) expressing a nucleic acid sequence according to the invention in acell, and ii) isolating the expressed antibody product. Additionally,the method may include iii) purifying the antibody.

Screening and Isolation of B Cells

Transformed B cells are screened for those producing antibodies of thedesired antigen specificity, and individual B cell clones can then beproduced from the positive cells.

The screening step may be carried out by ELISA, by staining of tissuesor cells (including transfected cells), a neutralization assay or one ofa number of other methods known in the art for identifying desiredantigen specificity. The assay may select on the basis of simple antigenrecognition, or may select on the additional basis of a desired functione.g. to select neutralizing antibodies rather than just antigen-bindingantibodies, to select antibodies that can change characteristics oftargeted cells, such as their signaling cascades, their shape, theirgrowth rate, their capability of influencing other cells, their responseto the influence by other cells or by other reagents or by a change inconditions, their differentiation status, etc.

The cloning step for separating individual clones from the mixture ofpositive cells may be carried out using limiting dilution,micromanipulation, single cell deposition by cell sorting or anothermethod known in the art. Preferably the cloning is carried out usinglimiting dilution.

The immortalized B cell clones of the invention can be used in variousways e.g. as a source of monoclonal antibodies, as a source of nucleicacid (DNA or mRNA) encoding a monoclonal antibody of interest, forresearch, etc.

The invention provides a composition comprising immortalized B memorylymphocytes, wherein the lymphocytes produce antibodies with highneutralizing potency specific for hCMV, and wherein the antibodies areproduced at ≧5 pg per cell per day. The invention also provides acomposition comprising clones of an immortalized B memory lymphocyte,wherein the clones produce a monoclonal antibody with a high affinityspecific for hCMV, and wherein the antibody is produced at ≧5 pg percell per day. Preferably said clones produce a monoclonal antibody witha high potency in neutralizing hCMV infection.

Exemplary immortalized B cell clones according to the invention are1F11, 2F4, 5A2, 9A11 and 6G4.

Further, cell lines expressing exemplary antibodies of the invention,1F11 and 2F4 were deposited with the Advanced Biotechnology Center(ABC), Largo Rossana Benzi 10, 16132 Genoa (Italy), under the terms ofthe Budapest Treaty, on Jul. 16, 2008. These deposits are provided forthe convenience of those skilled in the art and are neither an admissionthat such deposits are required to practice the invention nor thatequivalent embodiments are not within the skill of the art in view ofthe present disclosure. The public availability of these deposits is nota grant of a license to make, use or sell the deposited materials underthis or any other patents. The nucleic acid sequences of the depositedmaterials are incorporated in the present disclosure by reference andare controlling if in conflict with any sequence described herein.

Epitopes

As mentioned above, the antibodies of the invention can be used to mapthe epitopes to which they bind. The inventors have discovered thatantibodies 1F11, 2F4, 5A2 and 9A11, that neutralize hCMV infection ofendothelial cells, epithelial cells, retinal cells and dendritic cells,are directed towards an epitope determined by a combination of the hCMVproteins UL130 and UL131A. Although the inventors do not wish to bebound to any theory, it is believed that the antibodies 1F11, 2F4, 5A2and 9A11 bind to either a conformational epitope formed by these twoproteins, or bind to one of UL130 and UL131A, the other being requiredfor specificity.

In addition, the inventors have discovered that antibody 6G4 thatneutralizes hCMV infection of endothelial cells, epithelial cells,retinal cells, and dendritic cells is directed towards an epitopedetermined by a combination of hCMV proteins UL128, UL130 and UL131A.Although the inventors do not wish to be bound to any theory, it isbelieved that the antibody 6G4 binds to either a conformational epitopeformed by these three proteins, or binds to one or two of UL128, UL130and UL131A, the other(s) being required for specificity.

The epitopes recognized by these antibodies may have a number of uses.The epitopes and mimotopes in purified or synthetic form can be used toraise immune responses (i.e. as a vaccine, or for the production ofantibodies for other uses) or for screening patient serum for antibodiesthat immunoreact with the epitopes or mimotopes. Preferably, such anepitope or mimotope, or antigen comprising such an epitope or mimotopeis used as a vaccine for raising an immune response. The antibodies ofthe invention can also be used in a method to monitor the quality ofvaccines in particular to check that the antigen in a vaccine containsthe correct immunogenic epitope in the correct conformation.

The epitopes may also be useful in screening for ligands that bind tosaid epitopes. Such ligands preferably block the epitopes and so preventinfection. Such ligands are encompassed within the scope of theinvention.

Recombinant Expression

The immortalized memory B cells of the invention may also be used as asource of nucleic acid for the cloning of antibody genes for subsequentrecombinant expression. Expression from recombinant sources is morecommon for pharmaceutical purposes than expression from B cells orhybridomas e.g. for reasons of stability, reproducibility, culture ease,etc.

Thus the invention provides a method for preparing a recombinant cell,comprising the steps of: (i) obtaining one or more nucleic acids (e.g.heavy and/or light chain genes) from the B cell clone that encodes theantibody of interest; and (ii) inserting the nucleic acid into anexpression host in order to permit expression of the antibody ofinterest in that host.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone that encodes the antibody of interest; and (ii) using thesequence information from step (i) to prepare nucleic acid(s) forinserting into an expression host in order to permit expression of theantibody of interest in that host. The nucleic acid may, but need not,be manipulated between steps (i) and (ii) to introduce restrictionsites, to change codon usage, and/or to optimize transcription and/ortranslation regulatory sequences.

The invention also provides a method of preparing a recombinant cell,comprising the step of transforming a host cell with one or more nucleicacids that encode a monoclonal antibody of interest, wherein the nucleicacids are nucleic acids that were derived from an immortalized B cellclone of the invention. Thus the procedures for first preparing thenucleic acid(s) and then using it to transform a host cell can beperformed at different times by different people in different places(e.g. in different countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture techniques can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cellsare well known in the art e.g. see reference 37).

The expression host is preferably a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g. CHO cells, NSO cells,human cells such as PER.C6 [Crucell; reference 38] or HKB-11 [Bayer;references 39 & 40] cells, myeloma cells [41 & 42], etc.), as well asplant cells. Preferred expression hosts can glycosylate the antibody ofthe invention, particularly with carbohydrate structures that are notthemselves immunogenic in humans. Expression hosts that can grow inserum-free media are preferred. Expression hosts that can grow inculture without the presence of animal-derived products are preferred.

The expression host may be cultured to give a cell line.

The invention provides a method for preparing one or more nucleic acidmolecules (e.g. heavy and light chain genes) that encodes an antibody ofinterest, comprising the steps of: (i) preparing an immortalized B cellclone according to the invention; (ii) obtaining from the B cell clonenucleic acid that encodes the antibody of interest. The invention alsoprovides a method for obtaining a nucleic acid sequence that encodes anantibody of interest, comprising the steps of: (i) preparing animmortalized B cell clone according to the invention; (ii) sequencingnucleic acid from the B cell clone that encodes the antibody ofinterest.

The invention also provides a method of preparing nucleic acidmolecule(s) that encodes an antibody of interest, comprising the step ofobtaining the nucleic acid from a B cell clone that was obtained from atransformed B cell of the invention. Thus the procedures for firstobtaining the B cell clone and then preparing nucleic acid(s) from itcan be performed at very different times by different people indifferent places (e.g. in different countries).

The invention provides a method for preparing an antibody (e.g. forpharmaceutical use), comprising the steps of: (i) obtaining and/orsequencing one or more nucleic acids (e.g. heavy and light chain genes)from the selected B cell clone expressing the antibody of interest; (ii)inserting the nucleic acid(s) into or using the nucleic acid(s) toprepare an expression host that can express the antibody of interest;(iii) culturing or sub-culturing the expression host under conditionswhere the antibody of interest is expressed; and, optionally, (iv)purifying the antibody of the interest.

The invention also provides a method of preparing an antibody comprisingthe steps of: culturing or sub-culturing an expression host cellpopulation under conditions where the antibody of interest is expressedand, optionally, purifying the antibody of the interest, wherein saidexpression host cell population has been prepared by (i) providingnucleic acid(s) encoding a selected B cell the antibody of interest thatis produced by a population of B memory lymphocytes prepared asdescribed above, (ii) inserting the nucleic acid(s) into an expressionhost that can express the antibody of interest, and (iii) culturing orsub-culturing expression hosts comprising said inserted nucleic acids toproduce said expression host cell population. Thus the procedures forfirst preparing the recombinant expression host and then culturing it toexpress antibody can be performed at very different times by differentpeople in different places (e.g. in different countries).

Pharmaceutical Compositions

The use of antibodies as the active ingredient of pharmaceuticals is nowwidespread, including the products Herceptin™ (trastuzumab), Rituxan™,Campath™, Remicade™, ReoPro™ Mylotarg™, Zevalin™, Omalizumab, Synagis™(Palivizumab), Zenapax™ (daclizumab), etc.

The invention thus provides a pharmaceutical composition containing theantibodies of the invention and/or nucleic acid encoding such antibodiesand/or immortalized B cells that express such antibodies and/or theepitopes recognized by the antibodies of the invention. A pharmaceuticalcomposition may also contain a pharmaceutically acceptable carrier toallow administration. The carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolized macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Preferred forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilizing and/or dispersing agents.Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. It is preferred that the compositions areadapted for administration to human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Typically, the therapeutic compositionsmay be prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g. whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

Compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g. a lyophilised composition, like Synagis™ and Herceptin™,for reconstitution with sterile water containing a preservative). Thecomposition may be prepared for topical administration e.g. as anointment, cream or powder. The composition may be prepared for oraladministration e.g. as a tablet or capsule, as a spray, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as drops.The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a patient.For example, a lyophilized antibody can be provided in kit form withsterile water or a sterile buffer.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule, an antibody fragment or variants andderivatives thereof. As such, it will be susceptible to degradation inthe gastrointestinal tract. Thus, if the composition is to beadministered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, preferably between 6 and 8, and more preferably about 7.The pH may be maintained by the use of a buffer. The composition may besterile and/or pyrogen free. The composition may be isotonic withrespect to humans. Pharmaceutical compositions of the invention arepreferably supplied in hermetically-sealed containers.

Pharmaceutical compositions will include an effective amount of one ormore antibodies of the invention and/or one or more immortalized B cellsof the invention and/or a polypeptide comprising an epitope that bindsan antibody of the invention i.e. an amount that is sufficient to treat,ameliorate, or prevent a desired disease or condition, or to exhibit adetectable therapeutic effect. Therapeutic effects also includereduction in physical symptoms. The precise effective amount for anyparticular subject will depend upon their size and health, the natureand extent of the condition, and the therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation and is withinthe judgment of a clinician. For purposes of the present invention, aneffective dose will generally be from about 0.01 mg/kg to about 50mg/kg, or about 0.05 mg/kg to about 10 mg/kg of the compositions of thepresent invention in the individual to which it is administered. Knownantibody-based pharmaceuticals provide guidance in this respect e.g.Herceptin™ is administered by intravenous infusion of a 21 mg/mlsolution, with an initial loading dose of 4 mg/kg body weight and aweekly maintenance dose of 2mg/kg body weight; Rituxan™ is administeredweekly at 375 mg/m²; etc.

Compositions can include more than one (e.g. 2, 3, 4, 5, etc.) antibodyof the invention, particularly where such antibodies bind to differentantigens (or to different epitopes in the same antigen) to provide anadditive or synergistic therapeutic effect. For example, one antibodymay bind to a combination of the hCMV proteins UL130 and UL131A (orcomplex) while another may bind to gB. In a further example, oneantibody may bind to the combination of UL128, UL130 and UL131A whileanother may bind to epitopes in the hCMV proteins gB, gH, gL, gM, gN,gO, UL128, UL130, UL131A and combinations thereof. Thus, one antibodymay be targeted to the mechanism that mediates infection of fibroblasts,while the other antibody may be targeted to the mechanism that mediatesinfection of endothelial cells. For optimal clinical effect it may wellbe advantageous to address both mechanisms of hCMV infection andmaintenance.

Antibodies of the invention may be administered (either combined orseparately) with other therapeutics e.g. with chemotherapeuticcompounds, with radiotherapy, etc. Preferred therapeutic compoundsinclude anti-viral compounds such as ganciclovir, foscarnet andcidofovir. Such combination therapy provides an additive or synergisticimprovement in therapeutic efficacy relative to the individualtherapeutic agents when administered alone. The term “synergy” is usedto describe a combined effect of two or more active agents that isgreater than the sum of the individual effects of each respective activeagent. Thus, where the combined effect of two or more agents results in“synergistic inhibition” of an activity or process, it is intended thatthe inhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

Antibodies may be administered to those patients who have previouslyshown no response to treatment for hCMV infection, i.e. have been shownto be refractive to anti-hCMV treatment. Such treatment may includeprevious treatment with an anti-viral agent. This may be due to, forexample, infection with an anti-viral resistant strain of hCMV.

In compositions of the invention that include antibodies of theinvention, the antibodies preferably make up at least 50% by weight(e.g. 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the totalprotein in the composition. The antibodies are thus in purified form.

The invention provides a method of preparing a pharmaceutical,comprising the steps of: (i) preparing an antibody of the invention; and(ii) admixing the purified antibody with one or morepharmaceutically-acceptable carriers.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell of theinvention. Thus the procedures for first obtaining the monoclonalantibody and then preparing the pharmaceutical can be performed at verydifferent times by different people in different places (e.g. indifferent countries).

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) to asubject that encodes the monoclonal antibody (or active fragmentthereof) of interest, such that the nucleic acid can be expressed in thesubject in situ to provide a desired therapeutic effect. Suitable genetherapy and nucleic acid delivery vectors are known in the art.

Compositions of the invention may be immunogenic compositions, and aremore preferably vaccine compositions comprising an antigen comprising anepitope found on a combination of the hCMV proteins UL130 and UL131A.Alternative compositions may comprise (i) an antigen comprising anepitope found on a combination of the hCMV proteins UL130 and UL131A,and (ii) an antigen comprising an epitope found on the hCMV proteins gH,gB or a combination of gM and gN. In another embodiment, compositions ofthe invention may be immunogenic compositions, and may be vaccinecompositions comprising an antigen comprising an epitope found on acombination of hCMV proteins UL128, UL130 and UL131A. Alternativecompositions may comprise (i) an antigen comprising an epitope formed bya combination of the hCMV proteins UL128, UL130 and UL131A, and (ii) anantigen comprising an epitope found on the hCMV proteins gB, gH or acombination of gM and gN. Vaccines according to the invention may eitherbe prophylactic (i.e. to prevent infection) or therapeutic (i.e. totreat infection), but will typically be prophylactic.

Compositions may include an antimicrobial, particularly if packaged in amultiple dose format.

Compositions may comprise detergent e.g. a Tween (polysorbate), such asTween 80. Detergents are generally present at low levels e.g. <0.01%.

Compositions may include sodium salts (e.g. sodium chloride) to givetonicity. A concentration of 10±2 mg/ml NaCl is typical.

Compositions may comprise a sugar alcohol (e.g. mannitol) or adisaccharide (e.g. sucrose or trehalose) e.g. at around 15-30 mg/ml(e.g. 25 mg/ml), particularly if they are to be lyophilised or if theyinclude material which has been reconstituted from lyophilized material.The pH of a composition for lyophilisation may be adjusted to around 6.1prior to lyophilization.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. Preferably, one or more of the immunoregulatoryagents include(s) an adjuvant.

The epitope compositions of the invention will preferably elicit both acell mediated immune response as well as a humoral immune response inorder to effectively address a hCMV infection. This immune response willpreferably induce long lasting (e.g. neutralizing) antibodies and a cellmediated immunity that can quickly respond upon exposure to hCMV.

Medical Treatments and Uses

The antibodies, antibody derivative of the invention or antibodyfragments thereof may be used for the treatment of hCMV infection, forthe prevention of hCMV infection or for the diagnosis of hCMV infection.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be tissue samples taken from,for example, salivary glands, lung, liver, pancreas, kidney, ear, eye,placenta, alimentary tract, heart, ovaries, pituitary, adrenals,thyroid, brain or skin. The methods of diagnosis may also include thedetection of an antigen/antibody complex.

The invention therefore provides (i) an antibody, an antibody fragment,or variants and derivatives thereof according to the invention, (ii) animmortalized B cell clone according to the invention, (iii) an epitopecapable of binding one of 1F11 or 2F4, or 6G4 or (iv) an epitope capableof binding one of 5A2 or 9A11, for use in therapy or (v) a ligand,preferably an antibody, capable of binding an epitope that binds anantibody of the invention, e.g., 1F11, 2F4 or 6G4, for use in therapy.

Also provided is a method of treating a patient comprising administeringto that patient (i) an antibody according to the invention, (ii) anepitope capable of binding one of 1F11 or 2F4, or 6G4 or (iii) anepitope capable of binding one of 5A2 or 9A11, or (iv) a ligand,preferably an antibody, capable of binding an epitope capable of bindingone of 1F11, 2F4 or 6G4.

The invention also provides the use of (i) an antibody, an antibodyfragment, or variants and derivatives thereof according to theinvention, (ii) an immortalized B cell clone according to the invention,(iii) an epitope capable of binding one of 1F11 or 2F4, or 6G4, (iv) aligand, preferably an antibody, that binds to an epitope capable ofbinding one of 1F11 or 2F4 or 6G4, (v) an epitope capable of binding oneof 5A2 or 9A11, or 6G4, or (vi) a ligand, preferably an antibody, thatbinds to an epitope capable of binding one of 5A2 or 9A11, or 6G4 in themanufacture of a medicament for the prevention or treatment of hCMVinfection.

The invention provides a composition of the invention for use as amedicament. It also provides the use of an antibody of the inventionand/or a protein comprising an epitope to which such an antibody bindsin the manufacture of a medicament for treatment of a patient and/ordiagnosis in a patient. It also provides a method for treating a subjectand/or of performing diagnosis on a subject, comprising the step ofadministering to them a composition of the invention. The subject ispreferably a human. One way of checking efficacy of therapeutictreatment involves monitoring disease symptoms after administration ofthe composition of the invention. Treatment can be a single doseschedule or a multiple dose schedule. The invention is useful for thetreatment of hCMV infection.

Preferably, an antibody, immortalized B cell clone, epitope orcomposition according to the invention is administered to groups ofsubjects particularly at risk of or susceptible to hCMV infection. Suchsubjects groups include immunocompromised subjects, such as thosesuffering from HIV or undergoing immunosuppressive therapy, such astransplant patients.

Antibodies of the invention can be used in passive immunization.

Antibodies and fragments thereof as described in the present inventionmay also be used in a kit for the diagnosis of hCMV infection.

Epitopes capable of binding an antibody of the invention, e.g., themonoclonal antibody 1F11 or 2F4 or 6G4 may be used in a kit formonitoring the efficacy of vaccination procedures by detecting thepresence of protective anti-hCMV antibodies.

Epitopes capable of binding an antibody of the invention e.g., themonoclonal antibody 5A2 or 9A11 described in the present invention maybe used in a kit for monitoring the efficacy of vaccination proceduresby detecting the presence of protective anti-hCMV antibodies.Antibodies, antibody fragments or variants or derivatives thereof asdescribed in the present invention may also be used in a kit formonitoring vaccine manufacture with the desired immunogenicity.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing a monoclonal antibody with one or morepharmaceutically-acceptable carriers, wherein the monoclonal antibody isa monoclonal antibody that was obtained from an expression host of theinvention. Thus the procedures for first obtaining the monoclonalantibody (e.g. expressing it and/or purifying it) and then admixing itwith the pharmaceutical carrier(s) can be performed at very differenttimes by different people in different places (e.g. in differentcountries).

Starting with a transformed B cell of the invention, various steps ofculturing, sub-culturing, cloning, sub-cloning, sequencing, nucleic acidpreparation etc. can be performed in order to perpetuate the antibodyexpressed by the transformed B cell, with optional optimization at eachstep. In a preferred embodiment, the above methods further comprisetechniques of optimization (e.g. affinity maturation or optimization)applied to the nucleic acids encoding the antibody. The inventionencompasses all cells, nucleic acids, vectors, sequences, antibodiesetc. used and prepared during such steps.

In all these methods, the nucleic acid used in the expression host maybe manipulated to insert, delete or amend certain nucleic acidsequences. Changes from such manipulation include, but are not limitedto, changes to introduce restriction sites, to amend codon usage, to addor optimize transcription and/or translation regulatory sequences, etc.It is also possible to change the nucleic acid to alter the encodedamino acids. For example, it may be useful to introduce one or more(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, oneor more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid deletionsand/or one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acidinsertions into the antibody's amino acid sequence. Such point mutationscan modify effector functions, antigen-binding affinity,post-translational modifications, immunogenicity, etc., can introduceamino acids for the attachment of covalent groups (e.g. labels) or canintroduce tags (e.g. for purification purposes). Mutations can beintroduced in specific sites or can be introduced at random, followed byselection (e.g. molecular evolution). For instance, one or more nucleicacids encoding any of the CDR regions, heavy chain variable regions orlight chain variable regions of antibodies of the invention can berandomly or directionally mutated to introduce different properties inthe encoded amino acids. Such changes can be the result of an iterativeprocess wherein initial changes are retained and new changes at othernucleotide positions are introduced. Moreover, changes achieved inindependent steps may be combined. Different properties introduced intothe encoded amino acids may include, but are not limited to, enhancedaffinity,

General

The term “comprising” encompasses “including” as well as “consisting of”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a patient is intended toinclude prevention and prophylaxis. The term “patient” means all mammalsincluding humans. Examples of patients include humans, cows, dogs, cats,horses, goats, sheep, pigs, and rabbits. Preferably, the patient is ahuman.

The following are methods which can be used to practice the invention.

EXAMPLES Example 1: Cloning of B Cells and Screening for hCMVNeutralizing Activity

Two donors with high hCMV neutralizing antibody titers in the serum wereidentified. Memory B cells were isolated and immortalized using EBV andCpG as described in reference 36. Briefly, memory B cells were isolatedby negative selection using CD22 beads, followed by removal of IgM⁺,IgD⁺ IgA⁺ B cells using specific antibodies and cell sorting. The sortedcells (IgG⁺) were immortalized with EBV in the presence of CpG 2006 andirradiated allogeneic mononuclear cells. Replicate cultures eachcontaining 50 memory B cells were set up in twenty 96 well U bottomplates.

After two weeks the culture supernatants were collected and tested fortheir capacity to neutralize hCMV infection of either endothelial cells,epithelial cells or fibroblasts in separate assays. B cell clones wereisolated from positive polyclonal cultures as described in reference 36.IgG concentrations in the supernatant of selected clones were determinedusing an IgG-specific ELISA.

For the viral neutralization assay a titrated amount of a clinical hCMVisolate was mixed with an equal volume of culture supernatant or withdilutions of human sera containing neutralizing antibodies. After 1 hourincubation at room temperature the mixture was added to confluentmonolayers of either endothelial cells (e.g. HUVEC cells or HMEC-1cells), epithelial cells or fibroblasts in 96 well flat bottom platesand incubated at 37° C. for two days. The supernatant was discarded, thecells were fixed with cold methanol and stained with a mixture of mousemonoclonal antibodies to hCMV early antigens, followed by afluorescein-labeled goat anti mouse Ig. The plates were analyzed using afluorescence microscope. In the absence of neutralizing antibodies theinfected cells were 1,000/field, while in the presence of saturatingconcentrations of neutralizing antibodies the infection was completelyinhibited. The neutralizing titer is indicated as the concentration ofantibody (μg/ml) that gives a 50% reduction of hCMV infection.

Table 3 shows that three different types of antibodies have beenidentified. Those that can neutralize infection of fibroblasts, thosethat can neutralize infection of endothelial cells and those that canneutralize infection of both. This agrees with previous data thatdifferent proteins are responsible for tropism towards a particular celltype [7]. In addition to neutralization of endothelial cells, 1F11 and2F4 were observed to neutralize infection of epithelial cells, retinalcells and dendritic cells (data not shown).

TABLE 3 50% neutralization (μg/ml) Clone Specificity FibroblastsEndothelial cells 1F11 UL130/UL131A * 0.001 2F4 UL130/UL131A * 0.003 5A2UL130/UL131A * 0.002 9A11 UL130/UL131A * 0.001 7H3 gB 2 * 10C6 gB 0.30.3 5F1 gB 0.3 0.3 6B4 gB 0.5 2 Cytotec{circumflex over ( )} 5000 50Donor's Serum 33 1 *no neutralisation at the highest concentrationtested (i.e. >2 μg/ml). {circumflex over ( )}Cytotect (Biotest) is apool of hCMV hyperimmune IgG.

Some antibodies neutralized infection of both fibroblasts andendothelial cells at IgG concentrations ranging from 0.3 to 0.5 μg/ml.Other antibodies (1F11, 5A2, 9A11 and 2F4) failed to neutralize hCMVinfection of fibroblasts, but neutralized the infection of endothelialcells and did so at extremely low concentrations ranging from 0.001 to0.004 μg/ml (more than 1,000 fold more potent than previously knownantibodies capable of neutralizing infection of non-fibroblast cells).

Note that since the initial characterization, it has been determinedthat 5F1 binds to an epitope of gB rather than gH. This is consistentwith the results which demonstrate that blocking gB allowsneutralization of infection of fibroblasts as observed for 7H3, 1006 and6B4.

Table 4 shows that 6G4, which has been shown to be specific for acombination of the hCMV proteins UL128, UL130 and UL131A, was able toneutralize hCMV infection of endothelial cells, retinal cells anddendritic cells at very low concentrations (i.e. with high potency).

TABLE 4 50% neutralisation (μg/ml) Fibro- Endothelial/retinal/dendriticClone Specificity blasts cells 6G4 UL128/UL130/UL131A * 0.004Cytotec{circumflex over ( )} 5000 50 Donor's 33 1 Serum *noneutralisation at the highest concentration tested (i.e. >2 μg/ml).{circumflex over ( )}Cytotect (Biotest) is a pool of hCMV hyperimmuneIgG.

Example 2: Identification of the Target Antigens Recognized by theMonoclonal Antibodies

Human MRC-9 fibroblasts were infected with a clinical hCMV isolate.After 3 days the cells were metabolically labeled with ³⁵S Methionineand Cysteine. After preclearance of the lysate human monoclonalantibodies 1F11 and 2F4 were added and immunocomplexes were precipitatedby the addition of Protein A beads and resolved on SDS-PAGE (FIG. 1). Ahuman monoclonal IgG antibody with irrelevant specificity was used asnegative control. The results show that human monoclonal antibodies 1F11and 2F4 precipitate complexes of hCMV proteins.

To map the specificity of the human monoclonal antibodies, expressionvectors encoding hemagglutinin (HA)-tagged UL128Δ1-27, UL130Δ1-25 andUL131AΔ1-18 hCMV proteins lacking signal peptides were constructed.Vectors encoding full length hCMV proteins UL128, UL130, UL131A, gH, andgL were constructed as well. HEK293T cells were transfected with thesevectors alone or in combination. After 36 h, cells were fixed,permeabilized and stained with an anti-HA antibody (to control forefficiency of transfection) and with monoclonal antibodies followed by agoat anti-human IgG. A HuMab IgG with irrelevant specificity was used asnegative control. FIG. 2A shows that the human monoclonal antibodies1F11 and 2F4 recognize an epitope determined by a combination of thehCMV proteins UL130 and UL131A. FIG. 2B shows that the human monoclonalantibodies 5A2 and 9A11 recognize an epitope determined by a combinationof the hCMV proteins UL130 and UL131A. Antibody 6G4 stained cellsco-expressing at least full length UL128, UL130 and UL131A. Theintensity of the 6G4 staining was increased when gH and gL wereco-transfected together with UL128, UL130 and UL131A to reconstitute theputative whole glycoprotein complex gCIII (FIG. 6). These data suggestthat the monoclonal antibody 6G4 is specific for an epitope determinedby a combination of the hCMV proteins UL128, UL130 and UL131A (FIG. 6).Most likely this epitope is in the proper conformation only when UL128,UL130 and UL131A are assembled in gCIII with gH and gL.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

Conclusions

The above results define human monoclonal antibodies that are capable ofneutralizing with high potency and selectivity hCMV infection of humanendothelial cells and epithelial cells. To identify the epitoperecognized, the antibodies were tested for their capacity toimmunoprecipitate proteins from hCMV infected cells (FIG. 1). Humanmonoclonal antibodies 1F11 and 2F4 precipitated several proteins withapparent molecular weights of ˜15, 33-35 and ˜100 KDa. These patternsare compatible with the precipitation of a complex containing gH, gL andUL128, UL130 and possibly UL131A.

To better define the target of these antibodies we characterized theircapacity to stain HEK293T cells transfected with vectors encodingHA-tagged UL128, UL130 and UL131A. As shown in FIG. 2A, 1F11 and 2F4stained only cells coexpressing UL130 and UL131A, suggesting that theyrecognize a conformational epitope determined by a combination of thesetwo hCMV proteins. This conclusion is supported by the fact that theseantibodies do not react in a western blot with lysates of infected ortransfected cells run under reducing, denaturing, conditions (data notshown).

Similar results were observed for 5A2 and 9A11. FIG. 2b shows that theseantibodies stained only cells coexpressing UL130 and UL131A, suggestingthat they recognize a conformational epitope determined by a combinationof these two hCMV proteins. In addition, as shown in FIG. 6, antibody6G4 appears to be specific for a conformational epitope determined by acombination of the hCMV proteins UL128, UL130 and UL131A.

Example 3: Further Identification of the Target Antigens Recognized bythe Monoclonal Antibodies

To map specificities of human monoclonal antibodies neutralizinginfection of fibroblasts an expression vector encoding full length gBwas constructed. HEK293T cells were transfected with this vector. After36h, cells were fixed, permeabilized and stained with human monoclonalantibodies

(HuMab) followed by goat anti-human IgG. FIG. 4 shows that monoclonalantibodies 7H3, 1006, 5F1, and 6B4 (but not an IgG antibody of anirrelevant specificity) specifically stained cells transfected with gB,indicating that they recognize an epitope of gB. Of note, the monoclonalantibodies 1006, 5F1 and 6B4 neutralize infection of fibroblasts andendothelial cells, whereas the monoclonal antibody 7H3 neutralizes theinfection of fibroblasts (but not of endothelial cells). This notionsuggests that the monoclonal antibodies 1006, 5F1, and 6B4 bind to afunctional epitope of gB that is distinct from the epitope bound by themonoclonal antibody 7H3.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES (THE CONTENTS OF WHICH ARE HEREBY INCORPORATED BY REFERENCE)

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1. An isolated antibody, or an antigen binding fragment thereof, that isspecific for a complex comprising hCMV proteins UL128, UL130, and UL131Aand inhibits infection of endothelial cells, retinal cells, or dendriticcells by human cytomegalovirus (hCMV), wherein the concentration ofantibody required for 50% inhibition of hCMV is 0.1 μg/ml or less. 2.The antibody or fragment of claim 1, wherein the concentration ofantibody required for 50% inhibition of hCMV is 0.01 μg/ml or less. 3.The antibody or fragment of claim 1 which is specific for aconformational epitope formed by the proteins.
 4. The antibody orfragment of claim 1, comprising a variable region comprising an aminoacid sequence set forth in SEQ ID NOs: 63 or
 64. 5. The antibody orfragment of claim 1, wherein the antibody is a human antibody, amonoclonal antibody, a human monoclonal antibody, a single chainantibody, Fab, Fab′, F(ab′)₂, Fv or scFv.
 6. A composition comprisingthe antibody or fragment of claim 1, and a carrier.
 7. The compositionof claim 6 further comprising a second antibody, or antigen bindingfragment thereof, which inhibits hCMV infection.
 8. A method of treatinga subject diagnosed with hCMV infection, comprising administering aneffective amount of the composition of claim
 6. 9. A method ofinhibiting hCMV infection of a cell, comprising contacting the cell withthe antibody or fragment of claim 1.